Neuroprotective Role Of Edaravone In Aβ-induced Neurotoxicity In SH-SY5Y Cells

Background and objectiveAlzheimer’s disease (AD) is a severe neurodegenerative disorder. It ischaracterized by the abonormal extracellular accumulation of amyloid β peptide (Aβ)and subsequent neuronal cell damage. In degenerating neurons, excessiveautophagosomes are found, suggesting the incidence of autophagic neuronal damage.Edaravone, a well-characterized free radical scavenger, is known as a neuroprotectantwhich protects against neurodamage in various in vivo and in vitro experimentalparadigms. Nevertheless, its neuroprotective role against autophagic cell death in ADremains unclear. In this study, we aim to investigate the effects and regulatorymechanism of edaravone in Aβ-induced autophagic neuronal damage in SH-SY5Ycells.Methods1、The in vitro AD-like neurotoxic model was established by exposing SH-SY5Ycells to20μM of Aβ1-42. The neuroprotective effects were determined in cellspre-treated with100μM of edaravone.2、Cell viability was examined by MTT assay. Cell apoptosis was evaluatedusing Annexin V/PI double staining followed by flow cytometric analysis. Theintracellular reactive oxygen species (ROS) level was determined usingdihydro-ethidium staining. The formation and accumulation of autophagic vacuoleswas assessed by monodansylcadaverine (MDC) staining. The expressions ofautophagy-related proteins (LC3and Beclin1) and AMPK signal-related proteins(AMPK and p-AMPK) were evaluated by Western blotting. The influence ofautophagy on cell damage was further examined after knocking down LC3expressionin cells by RNA interference. Pharmacological stimulation and inhibition of AMPKsignal pathway by AMPK stimulator phenformin (10mM) and compound C (10μM)was conducted to determine the effects of AMPK pathway disturbance on cell viability. Besides, the effects of combined AMPK pathway interference withedaravone treatment were also studied.Results1、Aβ1-42triggered AMPK signaling pathway, and induced over-activation ofautophagy, leading to neurotoxicity, all of which could be efficiently reversed byedaravone pre-treatment.2、Silencing of LC3had no significant impacts on AMPK signaling pathway innormal cells. However, LC3deficiency greatly downregulated the Aβ1-42-inducedelevation of p-AMPK, and prevented the cell viability reduction induced by Aβ1-42.Compared with single treatment, edaravone combined with LC3silencing yieldedenhanced neuroprotection.3、 Phenformin activated AMPK signaling pathway, triggered autophagyactivation, and significantly suppressed cell viability in SH-SY5Y cells. Edaravonecould reverse the phenformin-induced reduction of cell viability by inhibiting AMPKand autophagy activation.4、Compound C inhibited the activation of AMPK signaling pathway, suppressedAβ1-42-induced autophagy activation, and remarkably prevented neurotoxicity inducedby Aβ1-42. In addition, combined administration of compound C and edaravoneresulted in promoted neuroprotection against Aβ1-42treatment possibly throughenhanced AMPK and autophagy inhibition.ConclusionOur findings proposed and demonstrated that edaravone efficiently suppressedAβ1-42-induced neurotoxicity in SH-SY5Y cells. The neuroprotective effects ofedaravone might be achieved by inhibiting AMPK signaling pathway and subsequentautophagy inhibition in cells.